The development of new biodefense therapeutics against anthrax and botulinum neurotoxins (BoNT) has heightened with the recent threat of these agents being used as biological weapons. Lethal factor (LF), a component of the anthrax tripartite exotoxin, cleaves mitogen-activated protein kinase kinases (MAPKK; 1-3) thus disrupting signal transduction leading to macrophage lysis (4). Following inhalation of anthrax spores, spores can adhere to alveolar macrophages and subsequently germinate. Bacteria migrate to lymph nodes where they rapidly multiply and excrete the anthrax exotoxin composed of protective antigen (PA; 83 kDa), LF, and calmodulin-activated edema factor adenylate cyclase (EF; 89 kDa). To exert its lethal effect, LF must enter inside the cell compartment. This is mediated by PA that binds to cellular receptors and, following its proteolytic activation by the furin-like proprotein convertases and the release of the N-terminal 20-kDa fragment, generates the mature PA protein (PA63). Finally, PA63 heptamerizes and binds both LF and EF. Following endocytosis of the resulting complexes, the engulfed molecules of LF and EF are liberated and exert their toxic action.
Botulinium neurotoxins, represent even more dreadful bioterrorism agents, because BoNTs may be delivered by aerosol (7, 8). With the increased use of BoNTs in physical/cosmetic ailments (8-13), the potential misuse of these toxins is worrisome (14). Currently, the only available treatment once BoNT has invaded the nervous system is critical care mechanical ventilation (14). However, the effects of internalized BoNTs can last for months (15) in which mechanical ventilation is an intolerable burden for those affected (14). Botulinum neurotoxins are composed of a heavy chain (HC) and a light chain (LC) which is connected by a disulfide bridge (16). The HC binds to neurons which transports the LC into the cytosol (17). The LC is a zinc metalloprotease, similar to anthrax that cleaves neuronal proteins involved in the neurotransmitter release. There are seven serotypes (BoNT A-G) involved in the cleavage of a component of the soluble NSF-ethylmaleimide-sensitive factor attachment protein receptor proteins (18), which mediates the exocytosis of acetylcholine into neuromuscular junctions (14). Both BoNT serotypes A and E cleave SNAP-25 [synaptosomal-associated protein (25 KDa)] (19), while serotypes B, D, F, and G cleave vesicle-associated membrane protein (20-23) and serotype C cleaves both SNAP25 and syntaxin 1 (14, 24).
The lethal action of anthrax toxin can be neutralized at several stages during its entry into the cell. In fact, it would be possible to inhibit PA63 processing, pore assembly or binding to receptor; moreover a successful therapeutic treatment could prevent LF or EF binding or their translocation into the cytosol (Sellman, B. R., Mourez, M., Collier, R. J. Science 292, 695-697 (2001)). Nevertheless inhibition of LF protease activity is still the most promising avenue for this harmful disease (Schwarze, S. R., Hruska, K. A., Dowdy, S. F. Trends Cell Biol. 10, 290-295 (2000)).
Inhibition of LF protease activity is believed to be a promising avenue for this harmful disease (Schwarze, S. R., Hruska, K. A., Dowdy, S. F. Trends Cell Biol. 10, 290-295 (2000)). Thus, a continuing need exists for compounds that inhibit lethal factor (LF) protease activity of anthrax toxin.